Solvent effects
The solvent often exerts a profound influence on the quality and shape of the spectrum. For example, many aromatic chromophores display vibrational fine structure in nonpolar solvents, whereas in more polar solvents this fine structure is absent because of solute–solvent interaction effects. A classic case is phenol and related compounds, which have different spectra in cyclohexane and in aqueous solution. In aqueous solutions, the pH exerts a profound effect on ionisable chromophores because of the differing extent of conjugation in the ionised and the non-ionised chromophore. This change of spectrum with pH and associated change in the ionic state of a molecule and conjugation is illustrated for phenobarbital and other barbituric acids. Note the shift in absorption to higher wavelengths as the conjugation is increased (increase in alternating double and single bonds in the molecule). The quality of spectral measurement is affected directly by the type and purity of the solvent used. Each solvent has a cut-off wave length (which corresponds to about 10% trans mittance) and this varies with solvent purity (Table 15.1). A solvent should not be used below its cut-off wavelength, even though reference cell compensation is employed, because of the greater risk of stray-light effects. The UV spectra of some solvents are illustrated in Fig. 1. The UV cut-off of solvents must be borne in mind when selecting solvents for HPLC mobile phases, particularly for analytes that absorb only in the low UV. A solvent should be selected with a lower UV cut-off than the absorption wavelength of the analyte.
Figure 1 UV absorption of solvents (HPLC-grade unless otherwise stated): (A) acetonitrile (far-UV grade); (B) methyl t-butyl ether; (C) acetonitrile; (D) 1–chlorobutane; (E) methylene chloride; (F) acetic acid (AR grade); (G) ethyl acetate; (H) acetone; (I) hexane; (J) isooctane; (K) methanol; (L) tetrahydrofuran; (M) chloroform; (N) diethylamine (AR grade).
Some cautionary comments may be appropriate at this point. It is better to use single- or double-distilled water and to avoid deionized water, which can be contaminated with absorptive fragments of ion-exchange resin or contain bacterial metabolites; these can contribute significantly to nonspecific absorption at low wavelengths. Ethanol is normally used as the 96% v/v strength, since dehydrated alcohol is usually contaminated with traces of benzene added to form the azeotropic mixture for distillation. Acetonitrile can vary noticeably in quality, depending on the supplier. The grade supplied for use in HPLC is usually to be recommended. Acetone, sometimes used to clean cells, is highly absorptive and not always easily removed, despite its volatility and aqueous solubility. Chloroform and carbon tetrachloride absorb strongly at about 250 nm and should therefore only be used for measurements at wavelengths above about 280 nm. Given the safety considerations of chlorinated solvents, use of these is best avoided if possible. Ether, although transparent down to 220 nm, presents particular problems because of its volatility (unstable standard solutions) and inflammability. Although absorptivity is considered to be relatively insensitive to temperature changes, organic solvents in general suffer from high temperature-coefficients of expansion, so that for ultimate precision a cell provided with a thermostat may be required.
